This is an application for a K08 award that outlines a 5-year training plan to facilitate Dr. James Lue's career development as an independent academic physician-scientist. Dr. Lue recently completed his clinical fellowship in pediatric gastroenterology at Stanford University and is establishing himself as a young investigator in stem cell biology research. This K08 award will provide Dr. Lue with the support necessary to accomplish the following training goals: (1) to become an expert in liver stem cell biology, (2) to become an expert in advanced techniques in transcriptional profiling and gene expression analysis, (3) to become an expert in manipulation of animal models for the study of hepatocyte and stem cell transplantation, and (4) to develop an independent physician-scientist career. To achieve these goals, Dr. Lue has assembled a mentoring team comprised of a primary mentor, Dr. Jeffrey Glenn, who is an internationally recognized authority on the molecular virology of hepatitis C virus (HCV), and has additional interests in the differentiation of liver stem cells, human hepatocyte repopulation in small animals, and engineered human liver tissues, and three co-mentors: Dr. Julie Baker, who specializes in the genomics of human definitive endoderm and the generation of hepatocytes from human embryonic stem cells;Dr. Gary Peltz, who specializes in the development of genetic mouse models for the study of human liver disease and drug metabolism;and Dr. Eric Sibley, who is a member of the Pediatrics Mentoring Program at Stanford University and will provide academic career advice and mentorship throughout the award period. Due to the vast shortage of organ donors, researchers have long been in search of alternatives to whole-liver transplantation. Therapies involving repopulation of the liver via cell transplantation have been evolving over the past two decades. Dr. Lue's long-term objective is to produce large numbers of functioning hepatocytes from stem cells so that therapies can be developed to treat patients with liver failure. This project will focus on improving the efficiency of derivation of hepatocytes from human embryonic stem cells (Aim 1) and adult mesenchymal stem cells (Aim 2). Dr. Lue plans to improve differentiation efficiency by priming these stem cells with specific transcription factors (TFs) that are important in normal liver development. Overexpression of these TFs will produce a more homogeneous and competent stem cell population that is more suitable for subsequent hepatic differentiation. Aim 3 will focus on hepatocyte transplantation and improving hepatocyte engraftment by exploiting a well-described homing system that is native to the liver. Fresh human hepatocytes, as well as the stem cell-derived hepatocytes from Aims 1 &2, will be modified to overexpress a specific homing receptor and intrasplenically injected into highly immunodeficient mice to study liver engraftment and function. This research will form the basis for an R01 grant application conducting research on novel methods of developing clinically useful hepatocytes from stem cells. PUBLIC HEALTH RELEVANCE: Close to 16,000 people are currently on the waiting list for a liver transplant in the United States, the demand for organs is more than double the supply, and over 1,500 people die each year on the waiting list (statistics from the United Network for Organ Sharing, unos.org). Stem cell therapy for the treatment of liver disease is a promising alternative to whole-organ transplantation, but still requires further research to improve safety and efficacy.